In general, to determine the genus, species, subspecies or strain groups of an organism, laborious and expensive biochemical and physiological procedures have been utilized. Various immunological techniques have been used to identify organisms serologically. Bacterial agglutination, immunofluorescence or immuno-precipitin assays have all been employed for this purpose. Bacterial agglutination only can be used to identify pure culture isolates, is generally a subjective qualitative assay and often relies upon subtle differences in antibody titers to differentiate organisms. Immuno-precipitin assays again only can analyze pure culture isolates. The preparation and characterization of materials are time-consuming and interpretations of the results are strictly qualitative, subjective decisions. Immunofluorescence (IF) can be used to identify both pure culture isolates and the presence of an organism in a complex mixture of bacteria. The nature of IF requires the availability of a fluorescence microscope, which, in itself, can limit the usefulness of the technique. Preparing and reviewing individual slides for reaction are tedious procedures. The results from the IF are essentially qualitative and subjective. Also, the sensitivity of the IF has been shown to be significantly less than enzyme-linked immune assays.
Bacteroides species are common etiologic agents in human anaerobic infections (1). The black-pigmented Bacteroides, including Bacteroides melaninogenicus subsp. intermedius, Bacteroides melaninogenicus subsp. melaninogenicus and Bacteroides gingivalis have been implicated as pathogens in human periodontal disease (2-5). Likewise, Bacteroides asaccharolyticus, which may be similar to B. gingivalis, has been shown to predominate in periodontal lesions of beagle dogs (6,7) and the primate, Macaca arctoides (8).
Serological identification of various species of B. melaninogenicus has been reported using immunofluorescent antibodies (IF) directed to capsular antigens (9, 10, 11) or to whole organisms (8, 12, 13). Similarly, using soluble antigen preparations in immunoelectrophoretic and gel diffusion analyses, Reed and coworkers (14) showed serological differences between oral and normal B. asaccharolyticus strains, as well as among other species of black-pigmented Bacteroides. Other workers, using cell-surface, outer-membrane complex antigens, identified serological differences among the Bacteroides with an enzyme immunoassay (15). However, in these studies, some cross-reactions among oral and nonoral Bacteroides species were found. In order to obtain monospecific antisera, cross-adsorptions were necessary. Lambe (12) showed that B. melaninogenicus subsp. intermedius, B. melaninogenicus subsp. melaninogenicus and B. melaninogenicus subsp. asaccharolyticus (now B. gingivalis) from humans could be serologically identified by IF. Also, recently fluorescentantibody kits, for detecting the B. fragilis (Fluorotec-F) and B. melaninogenicus (Fluorotec-M) groups, have been produced commercially (12). However, Mouton and colleagues (16) have shown that human B. gingivalis is not identified by either of these reagents, while the Fluorotec-M does react with both B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaminogenicus. The association of these types of organisms in sites of periodontal breakdown generally has been determined using a predominant cultivatable flora approach.
Thus, there exists a need for a rapid, simple and effective method for the identification of microorganisms, particularly the identification of microorganisms from the oral cavity in the presence of complex bacterial mixtures of microorganisms.